1. Field of the Invention
The present invention relates to methods and materials for protecting mammalian cells from injury due to intrinsic membrane lysis, oxidation and/or invasion by destructive agents. In particular the invention relates to materials and methods for treating against and/or prophylactically inhibiting the injury causation. Even more particularly, the invention relates to bioactive agents and the use thereof for treating or prophylactically inhibiting phospholipase mediated injury and/or injury due to oxidation. In a specific sense the invention provides agents for preventing and/or treating inflammation and cell destruction in mammalian tissue and for protection and preservation of biologic material such as food and tissue samples.
2. The Prior Art Environment
The base structure of all living organisms is the cell which is structurally defined by its membranous lipoprotein envelope. The membranous network that holds the cell together maintains the ionic balance and provides the receptors to hormones and neurotransmitters that enable a cell to interact with its environment. This is pertinent to interaction with neighboring cells which enable isolated cells, tissues, or whole organisms to survive as both independent units and as participants in cellular interactions, in vitro and in vivo. Nutritional, kinetic, electro-physiological, excretory, and reproductive mechanisms are mediated through the self renewal of the lipoprotein membranes that bind the cell, its nucleus and organelles into a functional whole.
The cell has a preordained life to live in accordance with the balance superimposed by the information provided by the nucleus and the environment. A cell has a date of conception and a circumstance mediated or preordained time to die. The dictates of certain circumstances, i.e., physiological stimuli or pathologic injury, prescribe the manner of death and the time of death of cells. Cell death and/or injury is the result of both intrinsic and extrinsic factors and is key to the fate of the larger organism of which cells are a critical and necessary part.
That which is good for the cell, that which maintains its capacity to respond to change to ionic fluxes that maintain membrane potential and to repair injury under normal conditions should be good for its host or supportive to in vitro biologic production (i.e., tissue culture, monoclonal antibody, enzyme or endocrine production of pertinence to biotechnology). The integrity of cell membranes which maintain ionic flux, and electro-physiologic and/or hormonal or messenger responses is the key to cellular functional survival and longevity. Repair and resistance to injury is a function of the maintenance of lipoprotein membrane integrity.
Factors which govern cell function, renewal, reproduction and death are controlled by their effects on the phospholipid/protein envelope or cytoskeleton. The cell membrane controls the cellular clocks and ionic fluxes which govern responsiveness and survival. Damage to phospholipid/proteins, with particular emphasis on lipid peroxidation, membrane oxidation and the action of, phospholipases, governs resistance to injury, repair and host responses to environmental change and ionic and osmotic integrity.
Pathological events in a host under clinical circumstances result in massive cellular insult, initiated or mediated by loss of membrane integrity. The events are mediated by "death triggers" which digest and destroy cell membrane and propagate an injury by producing a cascade of cell membrane changes. Similar events in tissue culture are vital to the biologic availability of cells and cell products while still permitting cells to possess the capacity to respond to their environments or each other. By interfering with the cascade of external and internal events involving membrane integrity and toxic changes which lead to cell death, injury can be prevented, modified or reversed. This has been a major role of anti-inflammatory agents in the past.
The most important presently used clinically effective anti-inflammatory drugs include the corticosteroids and the non-steroidal anti-inflammatory agents (NSAIAs). These drugs act to control inflammation and to minimize cell injury by regulating the breakdown of phospholipids or the action of the products of such breakdown leading to the formation of prostaglandins and leukotrienes which are produced in increased quantities in inflammation and promote cell dysfunction and injury.
In addition, recent studies have demonstrated that cellular and extra cellular phospholipases may be activated by the generation of oxygen free radicals. This can establish a "vicious cycle" as phospholipase activation can release free radicals which, in turn, activate more phospholipases. In this regard, free radicals are produced from fatty acids released by the action of phospholipases, which are converted to prostaglandins and leukotrienes. Fatty acids and free radicals are known to be prime mediators in the cascade of reactions that result in membrane injury, cell death and inflammation.
In addition to effects involving free radical formation, an additional role for phospholipases, particularly phospholipase A.sub.2 (PLA.sub.2), is that through their action promoting fatty acid release, as an example they produce arachidonic acid derivatives that promote potassium (K.sup.+) ion channel opening which governs the electrophysiologic and second messenger responses of the cell. Thus, phospholipase inhibitors can modulate cell responses to membrane stimulation governing cell function.
One of the hallmarks of inflammation and cell injury is the breakdown of cellular membrane phospholipid. Phospholipids are the major structural building blocks of the cell membrane; they give rise to the barrier-structural and functional properties of membranes and their integrity is crucial to normal cell responsiveness and function. Phospholipid changes in cell membrane integrity, particularly changes in fatty acids at the 2 position, alter the fluidity of cell membranes, their receptor availability and the leakiness or availability of cellular contents to the external environment. The breakdown of phospholipid membranes results. in "unraveling" or lysis of cells, or results in holes in the cell membrane, the disruption or enhancement of ion channels, or the loss of membrane bound receptors which destroys integrity and functional survival.
During inflammation, phospholipases, from whatever source, that are normally under the control of natural suppressor systems, are activated to degrade membrane phospholipid which, in turn, generates oxygen free radicals. A key enzyme which is activated in inflammation is phospholipase A.sub.2 (PLA.sub.2) which acts on phospholipids as enzyme targets to release free fatty acids. These fatty acids (i.e., arachidonate) released by PLA.sub.2 are converted to potent biologically active metabolites, prostaglandins and leukotrienes, with the concomitant generation of oxygen free radicals. These PLA.sub.2 products have effects on K ion channels and G proteins involved in second messenger cell responses involved in cell membrane homeostasis. These metabolites, fatty acids and free radicals, are powerful mediators of pathophysiology which propagate injury and cell death or permit the nidation, survival and growth of pathogens or tumor cells.
The role of phospholipases, particularly PLA.sub.2, as membrane targeted enzymes, make them veritable "death triggers" as the expression of their degradation activity results in further production of inflammatory mediators leading to further membrane injury which propagates damage within the cell itself or into adjacent tissue. Thus, the spread of injury from the initial site to contiguous or distant sites can be promoted by the activation and/or release of PLA.sub.2.
In addition to the intrinsic membrane-related tissue breakdown via the activation of PLA.sub.2, phospholipases, and particularly PLA.sub.2, are part of the normal defensive system of the body. PLA.sub.2 is found in particularly high levels in human white blood cells (WBCs): polymorphonuclear leukocytes (PMNs) and phagocytic cells. WBCs play a role in resisting infection, but when these cells are mobilized to ward off injury and infection, PLA.sub. 2 is released from adherent and circulating WBCs and produces local tissue necrosis which increases the extent of initial injury. In addition, WBCs adhere to blood vessel walls where they release enzymes such as PLA.sub.2. WBCs also generate free radicals and thus promote damage to the vascular endothelium, lung alveoli or to tissue sites contiguous with WBC infiltration or concentration. Where inflammation is found, WBCs are usually present in abundance and the WBCs adhere to vascular endothelium, with release and activation of PLA.sub.2 resulting in damage to vascular integrity during shock and ischemia. Thus, in spite of being a prime defensive system of the body against infection, WBCs can also damage the body by propagating injury and inflammation beyond their normal defensive role.
The classic description of inflammation is "redness and swelling with heat and pain (Celsus, 100 AD)." Inflammation has been defined as the reaction of irritated and damaged tissues which still retain vitality. Inflammation is a process which, at one level, can go on to cell death, tissue necrosis and scarring and at another level, inflammation can be resolved with a return to normalcy and no apparent injury or with minimal changes, i.e., pigmentation, fibrosis or tissue thickening with collagen formation related to healing and scarring. The process is dynamic, with cell death as one consequence, and recovery, healing and scarring as another. For inflammation to occur as a process, cells must retain their vitality. Dead or severely compromised cells do not respond to inflammatory reactions. Injury in inflammation can also relate to the late results of fibrosis and scarring with the loss of blood vessels, tissue elasticity and cosmetic quality.
Inflammation, while a normal process of the body's resistance to injury and infection, can become aberrant leading to propagated injury with extensive scarring, tissue death and/or the death of the organism. Within certain limits, the inflammatoryreaction is stereotyped and it cannot distinguish between those instances in which the process protects the host and those in which the host is harmed.
Microscopically, inflammation is characterized by vasodilation, vascular leakage, enhanced lymphatic flow, platelet vascular adherence and clumping and WBC infiltration and vascular adherence and Phagocytosis with slowing of blood flow, red cell aggregation resulting in the formation of blood clots. Clinically, these local phenomena can be associated with pain, fever and swelling which can lead to local tissue destruction (granulation, caseation and necrosis) healing or scarring or to systemic symptoms of pain, fever, shock (prostration) hypotension, leading to death or recovery.
Microscopically, inflammation has been described as related to: (1) atony of the muscle coat of the blood vessel wall; (2) increased resistance to blood flow related to friction and adhesiveness of blood elements (i.e., red blood cells, proteins, white blood cells, platelets); and (3) enhanced permeability, i.e., loss of red blood cells, white blood cells and blood fluid through the vascular wall.
In physiological terms, these are described as hyperemia, edema, blood stasis, thrombosis, and hemorrhage. Inflammation can be mediated by humoral substances produced by tissue elements or infectious agents or by changes in pH (acidity) or oxygen concentration. Clinically, pain, fever, malaise, muscle, arterial and visceral spasm as well as headache and confusion states can accompany inflammation for whatever primary cause.
The above events are often mediated by phospholipase activation, followed by fatty acid release and the formation of free radicals. These events can be endogenous to the matrix of the body, the supporting cells and tissues that are functionally or systemically integrated or related to specialized host defense mediating cells, i.e., induced by white blood cells or platelet activity which respond as part of the body's defenses and which release phospholipases or free radicals as part of their role in resistance to infection, or their place in the normal maintenance of coagulative or vascular integrity, i.e., the prevention of hemorrhage, thrombosis or ischemia.
Alternatively, phospholipase activity can relate to exogenous enzyme activity released from infecting pathologic organisms such as viruses, bacteria, Rickettsia, protozoa, or fungi which posses phospholipase as factors intrinsic to their infectious activity. In this regard, infecting organisms such as bacteria, viruses, Rickettsia, protozoa or their toxins can stimulate infected cells or the endogenous defense system to release phospholipases, i.e.., PLA.sub.2, which can act locally or at distance sites to produce inflammation or tissue damage. In the case of Naegleria, a pathogenic amoeba with affinity for the brain, destruction of brain membranes induced by phospholipases secreted by Naegleria can occur at sites in the brain distant from where the organism is localized.
In regard to intrinsic effects of PLA.sub.2, the same is produced in extremely high concentration by inflamed collagenous spinal discs where its localized action is associated with severe pain and muscle spasm, as part of low back and cervical cord injury resulting in acute or chronic discomfort.
Phospholipases released from infecting organisms or as a result of tissue injury can induce coagulation of blood. proteins, producing ischemic injury at sites contiguous or distant to the primary disease. In considering the action of phospholipases, it must be recognized that their pathologic effects can be both local, regional or systemic. This is governed by the phospholipase enzyme released, the level of albumin, natural inhibitors of enzyme action, and factors of diffusion, circulation and tissue vulnerability based on intrinsic inhibitors or the susceptibility of previously damaged or oxidized membranes or proteins to phospholipase action.
Inflammation is associated with trauma, infection and host defense reactions, i.e., fever, malaise and shock, related to direct bacterial or virus killing or associated immune responses. Immune responses can be both beneficial, protective or tissue damaging as can be seen in their being responsible for resistance or cure of infection, or on the other hand, capable of producing autoimmune phenomenon that results in allergy, i.e., asthma, urticaria, host versus graft disease, glomerular nephritis, rheumatic fever, lupus and rheumatoid arthritis.
In regard to the current treatment of inflammation, corticosteroids are effective anti-inflammatories, but must be used with caution clinically because they are powerful immunosuppressants and inhibitors of fibroblast activity necessary for wound and bone repair. In addition, corticosteroids are diabetogenic drugs and their toxic side effects involve interference with wound repair and bone matrix formation, and result in sodium retention, potassium loss and decreased resistance to infection. Corticosteroids also have effects on steroid formation, blood pressure, protein utilization, fat distribution, hair growth and body habitus. Alternatively, the clinically active NSAIAs, such as aspirin, indomethacin, ibuprofen, etc., work by inhibiting the conversion of free fatty acids to prostaglandins. The side effects of NSAIAs include gastric ulceration, kidney dysfunction and Reye's Syndrome, and metabolites of prostaglandin can be either damaging or protective to cells depending on the structure of the prostaglandin produced or utilized pharmacologically and the route of administration, cell or tissue effected. In addition to effects on inhibition of cyclooxygenase, some of the NSAIAs, including ibuprofen, indomethacin and meclofenamate, directly inhibit PLA.sub.2 activity in vitro,
As discussed previously, in conjunction with fatty acid release, as part of phospholipid cell membrane mediated injury produced by phospholipase activation, leukotrienes are generated. These leukotrienes produced from membrane phospholipid breakdown, damage tissue through direct toxic action, effects on ionic channels, and associated free radical formations or by indirect effects on vascular smooth muscle or vascular endothelial lining via platelet, WBC, endothelial (blood vessel lining) or smooth muscle constricting interactions.
Leukotrienes are responsible for smooth muscle constriction leading to bronchospasm and the asthmatic attacks seen in allergy or infectious asthma. Thus, there is an ongoing active search for leukotriene inhibitors for clinical application in the treatment of allergy, asthma and tissue injury and inflammation.
Because the phospholipase activated biochemical pathway for the formation of prostaglandins and leukotrienes derived from free fatty acids is branched, inhibition of one branch of this pathway, as with NSAIAs, can create an imbalance in these reactive metabolites. This imbalance may actually aggravate inflammation and promote cell injury as evidenced by the gastric ulceration side effects of NSAIA's, which, along with pH changes have intraperitoneal inflammatory effects.
Due to these adverse effects of both steroids and NSAIAs, there is currently much clinical medical interest in identifying phospholipase inhibiting agents that do not have steroidal side effects, but like corticosteroids modulate the first step leading to the production of injurious metabolites, fatty acids and free radicals.
Free radicals, produced by white blood cells, tissue injury or metabolic processes, are highly reactive chemical species which, in the case of tissue injury, are most often derived from respiratory oxygen. Oxygen, while necessary for energetics of life, is also a toxin which, as the chemically related superoxide, or as peroxides, can damage tissue instead of supporting it. Free radicals derived from oxygen are critical to damage produced by radiation, inflammation, ischemia (loss of blood supply) or through excess oxygen inhalation or exposure. As stated previously, free radicals are used bywhite blood cells to destroy infecting organisms, but can, under circumstances of shock, infection and ischemia, damage or destroy the tissue they were meant to protect.
Free radicals, induced by radiation, oxygen exposure, chemical agents (i.e., alkylating agents, dioxin, paraquat) or white blood cell reactions may be tissue damaging or important to mutational changes associated with aging, radiation or chemotherapy injury, the development of cancer and hyperimmune proliferative disease such as rheumatoid arthritis. In addition, these reactive chemical species can, through oxidation of proteins, enhance the vulnerability of proteins to protease digestion.
In the prior commonly assigned '941, '330, '739 and '408 applications identified above, it was disclosed that PGBx, a fatty acid polymer, is a specific PLA.sub.2 inhibitor and prevents the generation of free radicals. PGB.sub.x is capable of inhibiting inflammation induced by PLA.sub.2 ; prevents arachidonate release from human PMNs and vascular endothelium and phospholipids; blocks the synthesis of eicosanoid prostaglandin precursors; protects lipids from auto-oxidation; and decreases endogenous lipid peroxide formation and oxidation of tissue homogenates.
The PGB.sub.x compounds delay aging in houseflies, prolong survival and decrease the age-related pigment in cardiomyocytes, protect paramecia from benzpyrene photoactivation lysis, block the production of superoxide from PMN WBCS, protect the myocardium or myocardial cells from anthracycline or high oxygen tension pro-oxidant injury, block carrageenin and adjuvant induced inflammation and arthritis, block platelet aggregation and blood clotting, and interfere with cytotoxic immune response; block IL-3 leukokine stimulation of mast cell proliferation in vitro, and spontaneous gamma interferon C2 complement release. These compounds can screen out cytotoxic ultraviolet exposure and are systematically and orally active and stable to autoclaving and filtration sterilization.
The general chemical structure of the PGB.sub.x and other oligomers disclosed in the above-identified '330 application has been hypothesized; however, the total defined chemistry required for satisfying regulatory agencies involved in drug development has remained obscure. Accordingly, what is still needed are pharmacologically active materials that are free of compounds having isomeric and/or structural variations.